25 - Ras and Raf: Anti-Cancer Drugs

Question 1

What happens when a Ras receptor is activated?

Answer 1

When a receptor is activated it dimerises leading to recruitment of Ras2 and SOS from the cytoplasm, recruitment of SOS allows for access to Ras which is tethered to the cell membrane by a lipid anchor, when SOS is recruited it interacted with the Ras switch domains allowing the switch from GDP to GTP

Question 2

When can Ras interact with Raf?

Answer 2

In GTP bound from the Ras can interact with downstream effectors including Raf

Question 3

What happens when Raf is activated?

Answer 3

Raf can be activated which leads to activation of the ERK MAP kinase pathway and transmission to the nucleus of a signal leading to phosphorylation of transcription factors like Elk involved in signal transduction pathways and cell proliferation

Question 4

How is Raf activated by Ras?

Answer 4

The Raf is not directly activated by Ras, but Raf is recruited to the cell membrane by active Ras
o At the cell membrane unknown kinases phosphorylate Raf and this triggers the activation of the ERK MAP kinase cascade

Question 5

Ras-Raf interaction

Answer 5

• Activated as interacts with the amino terminal regulatory region of Raf-1 and this leads to Raf-1 activation
• The purpose of Ras-Raf interaction may be to translocate Raf to the plasma membrane for activation

Question 6

What does activation of Ras engage?

Answer 6

Activation of Ras engages a phosphorylation cascade from Raf-1 to MEK (MAP kinase kinase) to the MAP kinase family

Question 7

What is prenylation?

Answer 7

Prenylation refers to linking of “isoprene” based groups
o Always Cys of CAAX (C-Cys), A= Aliphatic amino acids, X= aby residue)

Question 8

What do isoprene groups include?

Answer 8

Isoprene groups include:
o Farnesyl (15-carbon, three double bond)
o Geranylgeranyl (20-carbon, four double bond)

Question 9

What is the C-terminal?

Answer 9

The C-terminal region of small GTPases, is post-translationally modified with lipids
- In their C-terminal they all have a cysteine which is modified by isoprene groups
* Rap is closely related to Ras
* Rap, Rab and Rab and have GG groups

Question 10

What makes Ras different from other small G-proteins?

Answer 10

In Ras we have F linkages, making it different from other small G-proteins

Question 11

What happens when there is a farnesyl group that is not presenting there small G-proteins?

Answer 11

If we have a farnesyl group that is not presenting other small G-proteins and we block the attachment of the farnesyl group to Ras then we can make a drug that specifically prevents Ras from associating with the cell membrane without affecting the distribution of other small G-proteins

Question 12

Farnesyl farnesylation

Answer 12

Farnesyl: the sulphur of the cysteine has been covalently modified to add the farnesyl group

Question 13

Geranyl geranylation

Answer 13

Geranyl: longer with double bonds, attached by a covalent sulfur thiol ether bond in the C-terminus of the protein

Question 14

What is required for Ras proteins to be functional?

Answer 14

To be functional, the Ras proteins require post-translational lipid modificiation, translocation, and attachment to the plasma membrane

Question 15

Why were isoprenoid inhibitors invented?

Answer 15

Isoprenoid inhibitors, such as farnesyl transferase inhibitors (FTI), have been developed as anti-Ras agents for the treatment of many types of solid tumours whose proliferation is dependent on Ras

Question 16

What do isoprenoid inhibitor drugs do?

Answer 16

These drugs prevent Ras association with the plasma membrane thereby inhibiting coupling to mitogenic signalling cascades, such as the ERK pathway
* Currently these are in phase III clinical trials

Question 17

Give an example of a farnesyltransferase inhibitor

Answer 17

Tipifarnib (trade name Zarnestra) is a farnesyltransferase inhibitor that is being investigated in patients 65 yars of age and older with newly diagnosed acute myeloid leukemia (AML)

Question 18

What is targeted in the treatment of melanoma?

Answer 18

Raf is targeted for the treatment of melanoma

Question 19

How is Raf held in an OFF position?

Answer 19

Raf is held in an off position in the cytoplasm until there is RTK activation
* Its been held in an inactive conformation by interaction with 14-3-3 proteins which are proteins that interact with phosphylated serine and threonine residues

Question 20

What is the first step in the activation of Raf?

Answer 20

The first step in the activation of Raf in complex and involves dephosphorylation of Raf by serine/threonine phosphatases PP1 and PP2A
o These cause the phosphorylation of Raf and a particular residue and this loosens the association of the 14-3-3 protein but the Raf is still not fully active yet

Question 21

What does full activation of the Raf require?

Answer 21

Full activation of the Raf requires recruitment to the cell membrane by Ras in the GTP bound form, this interacts with the binding domain which displaces from the inhibitory binding to Raf allowing further dephosphorylation of Raf and then rephosphorylation by unknown kinases including probably Src

Question 22

What are melanocytes?

Answer 22

Melanocytes are specialised pigment cells that are found in the skin

Question 23

Stimulation of what induces melanocyte differentiation?

Answer 23

Stimulation of the melanocortin 1 receptor by a MSH (melanocyte stimulating hormone) stimulates cAMP production and this induces melanocyte differentiation

Question 24

What can MSH stimulate?

Answer 24

MSH can stimulate melanocyte proliferation in vitro, but only when combined with signals from RTKs

Question 25

How can we get a full mitogenic response?

Answer 25

Generally, RTK stimulate the activation of the ERK cascade but in these cells the stimulation is rather weak, so we don’t get a mitogenic response
* In this particular case to get a full mitogenic response we need to have some kind of crosstalk between the MSH signalling pathway and the RTK signalling pathway

Question 26

What is involved in the crosstalk between the MSH pathway and the RTK pathway?

Answer 26

We have a Gs coupled receptor stimulating the production of cAMP which normally stimulates melanogenesis
* However, proliferation of melanocytes is triggered by PKA stimulating the activation of B-RAF and that activates the ERK MAP kinase pathway

Question 27

What controls the crosstalk between the MSH pathway and the RTK pathway?

Answer 27

This is normally controlled by MSH- what happens in melanoma is that the BRAF becomes mutated and doesn’t need any input from the MC1R receptor and no longer needs cAMP
o Override this control leading to uncontrolled proliferation

Question 28

How long does it take to develop a drug?

Answer 28

Drug development is pretty slow and it can take 20 years to develop a drug

Question 29

What is BRAF?

Answer 29

BRAF became known as a drug target through the use of next generation sequencing

Question 30

What is known about CRAF?

Answer 30

Traditionally, with Raf signalling and ERK MAP kinase signaling researchers concentrated of CRAF, which I inhibited by PKA, and lots was known about their mechanisms of action
* With the understanding the mechanisms of action of CRAF it was found that those isoforms of Raf weren’t really involved in cancer and weren’t mutated very much

Question 31

What did next-generation sequencing reveal about BRAF?

Answer 31

Next generation sequencing revealed that by sequencing the BRAF gene there were lots of variations of mutants of BRAF associated with various cancers so mutations of BRAF was found to occur with 60% of melanoma cases and also with other types of cancers like thyroid or ovarian

Question 32

What did next generation sequencing of BRAD lead to?

Answer 32

This led to the realisation that BRAF was directly linked to cancer and within 10 year, people have devised small molecules to inhibit Raf activity, and these were translated into the clinic and used to treat melanoma
* They found mutations often occurred near the activation segment- amino acids 600 was often mutated in melanoma and other cancers

Question 33

What do BRAF mutations correspond to?

Answer 33

• 90% of these mutations correspond to the amino acid substitution V600E
• Many of the other 20% accounts for missense mutations in the activation segment near the V600

Question 34

What is the V600E mutation?

Answer 34

The V600E mutation is oncogenic because it mimics the phosphorylation of T599 and/or S602 in the activation segment and so B-RAF becomes constitutively in a Ras independent manner

Question 35

What are 3 features of the V600E BRAF

Answer 35

1. V600EBRAF is a founder mutation
   
   • Detected in human premalignant melanocytic navi and colorectal hyperpleastic polyps
   • In mice, V600EBRAF induces naevi and hyperplastic crypts
2. V600EBRAF is a driver mutation
   
   • Transforms cells in culture
   • Induces malignant tumours in mice
3. V600EBRAFis a validated therapeutic target
   
   • siRNA studies show tumours are addicted to V600EBRAF
   • inhibitors for V600EBRAF proving effective in clinic (PLX4032)

Question 36

Give an example of other drug targets that are being developed?

Answer 36

Other drug targets are being developed-for example a couple of farnesyl transferase inhibitors have been developed which block Ras activation by receptor activation

Question 37

What strategies are there for inhibiting Ras/Raf signalling?

Answer 37

• PLX4032 as a Raf inhibitor
• MEK inhibitors - there is a range being developed and none are being used in the clinic because if we inhibit MEK activity the cells tend to develop resistance by upregulating other pathways
• No Erk inhibitors
• Ras is known to activate the PI3K pathway, activating ATT (PKD) and downstream targets
• mTOR inhibitors are being tested
• PKD inhibitors and PI3K inhibitors are being tested